The ascomycetes comprise a class of fungi which includes among its members a number of useful, familiar, and important eucaryotic organisms. Perhpas the best known among these are the yeast strains which are familiar to all in connection with production of alcoholic beverages and of bakery goods. Other well known representatives include Neurospora crassa which is most frequently encountered as a pinkish bread mold, the ringworm which accounts for a formerly more common skin disease, and the blue molds which account for the flavor and appearance of bleu and Roquefort cheese.
The ascomycetes occur in two significantly distinctive types: those which form a filamentous mycelium, the "euascomycetes" and those which do not, the "hemiascomycetes". One authoritative taxonomy of the ascomycetes (never a static classification, however) sets forth five subclasses, four of which are of the first subtype, and one of which is of the second. (Alexopolous, C. et al, Introductory Mycology (J. Wiley and Sons, N.Y.).
The subclass hemiascomycetidae, which is of the second type, includes the yeasts such as Saccharomyces and Candida. Genetic manipulation of members of this subclass and methods for transformation thereof, have been relatively well developed (see, for example, Van Solingen, P., et al, J Bact (1977) 130:946). Of the remaining subclasses, two contain organisms of industsrial importance: the Plectomycetidae which contains black molds, blue molds, and ringworms, including the various Aspergillus and Penicillium species; and the subclass Pyrenomycetes which is represented by the various organisms causing mildew and by N. crassa, and by strains of Trichoderma and of Cephalosporium. While among the representatives of these two filamentous subclasses there are found a wide variety of industrial organisms used for, for example, the production of primary metabolites such as citric acid, of secondary metabolites such as antibiotics, most notably penicillin and cephalosporin and various industrial proteins and enzymes such as proteases and other hydrolytic enzymes, relatively little has been done with respect to the genetics of these organisms. In general they have proved refractory to transformation and to genetic manipulation. Recently, a transformation system has been developed for N. crassa (Case, M E., et al, Proc Natl Acad Sci (USA) (1979) 76:5259). However, vectors useful for general genetic manipulation even in this species have not been disclosed. More recently, Ballance, D. J., et al, Biochem Biophys Res Comm (1983) 112:284 succeeded in transforming Aspergillus nidulans (a member of the Plectomycetidae subclass) with a segment of DNA carrying the genes for orotidine-5'-phosphate decarboxylase derived from N. crassa (a member of the Pryrenomycetes subclass). However, as the N. crassa gene is from a species foreign to the host, the DNA of the transforming plasmid does not integrate into the host genome at a site complementary to an indigenous gene, but rather integrates at a random location, and with relatively low frequency. Also, Tilburn, J et al, Gene (19) 26:205 have transformed A. nidulans with a plasmid carrying the amdS A. nidulans gene. However, this gene, which is expressed to confer phenotypic characteristics including sensitivity to fluoroacetate and ability to grow on acetamide, has been found only in A. nidulans strains, and thus may find no indigenous counterparts in the remaining filamentous ascomycetes species.
There is, thus, no currently available system for providing a broad spectrum of tools for genetic manipulation of the important members of the filamentous ascomycetes sublcasses; members such as Aspergillus nidulans, Aspergillus niger, plant pathogens, such as Alternaria and Fusarium, and the various Penicillium species, and Cephalosporium species. The present invention provides such systems and tools which enable genetic modification of industrially important strains to perform conversion processes of which they were previously incapable, production and secretion of large quantities of desired foreign proteins by these organisms, and in general, addition of the filamentous ascomycetes to the arsenal of useful recombinant host organisms and gene sources available to the biotechnologist.